Title

Author

Graduation Date

Spring 5-5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Parmender P. Mehta

Abstract

Gap junctions (GJ)s are conglomeration of several cell-cell channels at cell-to-cell contact sites involved in the direct intercellular exchange of small growth regulatory molecules. Defects in assembly of GJ-forming proteins, called connexins (Cxs), are observed in many cancers, yet the molecular basis of this defect remains unknown. Connexin32 (Cx32) is expressed by the polarized cells in epithelia. The carboxyl-terminal tail (CT) of Cx32, although not required to initiate GJ formation, orchestrates several aspects of GJ dynamics, function and growth. Our studies have discovered that the CT of Cx32 harbors a tyrosine-based [YXXØ]-type and two dileucine-based [DE]XXXL[LI]-type motifs, which govern the intracellular sorting and endocytosis of transmembrane proteins. We explored their role in regulating endocytosis and GJ-forming ability of Cx32. One dileucine motif, designated as LI, was located near the juxtamembrane domain, whereas another, designated as LL, was located distally. We also discovered a non-canonical motif, designated as LR. Our results showed that the LL and LR motifs regulated the endocytosis of Cx32 by the clathrin-mediated pathway. Rendering the LI motif nonfunctional inhibited GJ assembly by augmenting Cx32 endocytosis via the LL and LR motifs. We also found that the tyrosine-based motif, acted as a possible endocytic motif. Moreover, our studies showed that the LI motif regulates basolateral sorting of Cx32 from the Golgi to the cell surface in polarized cells only.

We also found that the CT of Cx32 harbored three cysteines. These cysteines were likely to be modified by palmitoylation, and two of them were part of a CAAX box motif, known to be modified by prenylation. Our studies have shown for the first time that cysteine 217 of Cx32 was palmitoylated. However, we found that mutating these cysteines singly affected neither the trafficking nor the ability of Cx32 to assemble into GJs. Intriguingly, we discovered that mutating all cysteines together or cysteine 280 and 283 in combination, blocked the transport of Cx32 from the Golgi to the cell surface. Overall, our studies here show that the trafficking, endocytosis, and assembly of Cx32 are modulated in a complex manner by different regulatory motifs found on its CT.